Project Summary Endocytosis is the process of uptake of cargo and ?uid from the extracellular space to inside the cell; defects in endo- cytosis contribute to a wide spectrum of diseases including cancer, neurodegeneration, and heart disease. Clathrin- mediated endocytosis (CME) is an archetypal example of a membrane deformation process where multiple variables such as pre-existing membrane curvature, membrane bending due to the protein machinery, membrane tension regula- tion, and actin-mediated forces govern the progression of vesiculation. Advances in imaging technology have recently led to an explosion in morphological and biochemical data sets that track the progression of CME. While computa- tional modeling of lipid bilayers has provided insight into the mechanics of membranes in general, a mechanistic and predictive framework that can relate the plasma membrane composition and plasma membrane-cytoskeleton interac- tions to the progression and robustness of CME is missing, resulting in a gap between the experimental advances in the study of CME and a predictive, mechanistic framework for harnessing CME for nanomedicines. Preliminary data from our group has shown that membrane tension plays an important role in governing the progression of CME. How does membrane tension govern the progression of CME in the presence of membrane-protein interactions and membrane-cytoskeleton interactions? Substantial preliminary data in this application supports the working hypothesis that membrane tension is a dynamic quantity that evolves over the progression of CME to modulate the energy bar- rier associated with vesiculation. Speci?cally, the work of the principal investigator, supported by ?ndings from others has identi?ed that membrane tension governs CME through a snapthrough instability. Building on these preliminary ?ndings, the goal of the proposed work is to elucidate the fundamental biophysical principles of CME. In the proposed work, we have outlined three hypotheses and aims aims that will enable us to close this knowledge gap. Aim 1 will test the hypothesis that membrane-protein interactions during CME are regulated by membrane tension dynamically; this hypothesis will be tested using new theoretical and computational models that will incorporate the energetics of mem- brane-protein interactions and in-plane diffusion of proteins along the membrane. It is expected that membrane tension will emerge as a dynamic modulator of local membrane deformations due to protein interactions. Aim 2 will test the hypothesis that force generation during CME depends on the actin organization around an endocytic pit; this hypoth- esis will focus on the development of theoretical models that incorporate the dynamic and stochastic actin-membrane interactions and predict the spatio-temporal organization of actin ?laments around an endocytic pit. Aim 3 will test the hypothesis that pre-existing curvature of the membrane can modify the energy landscape of the progression of CME; models will be developed to test this hypothesis using different initial curvatures of the substrate. Collectively, the insights provided by the modeling effort conducted in these three aims will provide insight into how membrane-protein and membrane-cytoskeleton interactions affect the progression of CME.